Histamine is a vitally important messenger in the human body: In the periphery, it regulates vascular permeability, vasoconstriction, inflammation, and acid secretion by the stomach; in the brain it is a neurotransmitter employed by hypothalamic cells that project widely throughout the brain and spinal cord, and even to the retina. While histamine receptors are well known, a histamine transporter has been elusive. We propose to clone and study histamine transporters from invertebrate photoreceptors, neurons that clearly and specifically take up histamine. We will begin with Drosophila (fly). Candidate sequences will be identified by screening the Drosophila database, then will be further screened for their presence in photoreceptors of Drosophila compound eyes using in situ hybridization. Positive results will be followed by obtaining the full length clone using the PCR reaction, converting the cDNA to cRNA, injecting it into oocytes, and assaying for both Na-dependent (3H)histamine uptake and histamine-induced currents. Alternatively, we will pursue initial results using cDNA libraries from the eyes of Balanus (barnacle) and Limulus (horseshoe crab). Full-length candidate genes, identified using PCR amplification of sequences from the libraries, will be injected into oocytes for assay of histamine transport. Another alternative is injection of barnacle or fly mRNA into oocytes followed by expression cloning. Successful cloning of an invertebrate histamine transporter will be followed by attempt to clone a possible mammalian homologue. Cloned histamine transporters, expressed in Xenopus oocytes, will be characterized using electrophysiological techniques. The substrate selectivity of these carriers will be determined. The substrate and ionic dependence of the carriers will be defined which should permit understanding of how these transporters control the extracellular substrate concentration. This is of particular interest in the case of the photoreceptors, where the extracellular histamine concentration is thought to be continually maintained in a range that activates postsynaptic receptors. Determining the voltage dependence of the transporter should provide greater understanding of the observation (in barnacle photoreceptors) that histamine uptake is greater with depolarization. The pharmacology of each transporter will also be described, with special attention to drugs known to affect mental function. Successful cloning of a mammalian homologue would open the door to localizing this transporter within regions and cell types of the brain and determining whether it is present in other histamine-containing cells of the body.